Testing for potentially harmful microbes in both natural and human-modified environments represents a significant public health challenge. Microbial contamination of waters can lead to numerous serious issues. Bacterial contamination can cause adverse economic impacts for the affected areas by closing popular recreational areas for extended periods of time. This is particularly relevant in the heavily visited recreational waters and the swimming beaches in urban centers. In addition, bacterial contamination can cause severe illnesses such as gastrointestinal disorders. Microbial contaminants may not be limited to surface or recreational waters. In fact, a far more costly migration of these microbial contaminants is their movement to groundwater supplies which are used for drinking water.
The cysts of Cryptosporidium are of increasing importance because of their presence in water supplies. When in the gut, four spindle-shaped motile sporozooites burst from the cyst to infect gut epithelial cells and continue their life cycle. Entamoeba histolytica, another water-borne pathogen, can cause diarrhea or a more serious invasive liver abscess. When in contact with human cells, these amebae are cytotoxic. Giardia is found in contaminated rivers and lakes and is also be contracted via contaminated foods. There is some evidence that a heavy infection of attached Giardia physically blocks the important transport of nutrients across the epithelium (see, for example, www.cellsalive.com/parasit.htm).
Detection of airborne pathogens has also garnered much attention in light of the recent anthrax attack where anthrax spores were disseminated from mail packages.
A recent news report has stated that the anthrax mailed to a Senate office last fall was able to become airborne again even after it settled in the office. The fact that ordinary movement in the office was enough to send anthrax back into the air provides evidence that the spores were altered to make them more dangerous. Scientists thought that once the anthrax-laden envelope was opened, the spores would settle in the office and would be unlikely to become airborne again. But simulation of normal workplace activities such as paper handling, walking around the office and mail sorting several weeks after the envelope arrived, caused the spores to become airborne. Measurements of anthrax in the air of the office were substantially higher after researchers simulated everyday office activity than when the office was more still. And more than 80% of the airborne anthrax spores were of a size that could easily be breathed into the lungs (see, for example, www.ph.ucla.edu/epi/bioter/officeactivityanthrax.html).
According to a study published in 1999 by the Centers for Disease Control and Prevention (Atlanta), there were almost 14 million cases of foodborne illness caused by known pathogenic microbes (bacteria, parasites, and viruses) in the United States in 1997, and these illnesses caused more than 60,000 hospitalizations and almost 2000 deaths (1). Although ˜70% of the reported illnesses were caused by food contaminated with Norwalk-like viruses, almost half of the deaths were the result of infection with Listeria monocytogenes and Salmonella species bacteria. Foodborne microbial contamination comes in a variety of guises and from myriad sources. Perhaps the best-known foodborne illnesses come from the various subspecies of E. coli. Largely associated with raw or undercooked ground beef, E coli. infection leads to gastrointestinal disorders. Infection is usually self-limited and can last for about eight days. In some cases, however, infection can lead to hemolytic uremic syndrome, which causes renal failure and anemia, and it can be deadly to children, the elderly, and those with weakened immune systems. Certain subtypes of E. coli can cause severe, even lethal illness. For example, in the spring of 1992–93, a multi-state outbreak of over 500 cases of E. coli O157:H7 infections was associated with a restaurant chain (www.dhss.state.mo.us/MoEpi/moepi161.pdf).
One of the least understood but increasingly prevalent illnesses is caused by Listeria monocytogenes. Associated with raw milk, soft cheeses, and raw meats, L. monocytogenes infection can lead to meningitis, encephalitis, and intrauterine or cervical infections in pregnant women that can cause spontaneous abortion or stillbirth. Listeria is able to grow under a wide variety of conditions, including refrigeration.
Another common infection is caused by Salmonella species. Associated with raw meats, poultry, and eggs, Salmonella infection can lead to nausea, vomiting, cramps, and diarrhea. Typically, acute symptoms subside after a couple of days. In severe cases of S. typhi or paratyphi, however, infection can cause a typhoid-like fever and possible septicemia.
As with many other bacteria, Campylobacter jejuni infections also lead to gastrointestinal disorders. Infection is usually self-limiting and lasts 7–10 days. Associated largely with poultry, Campylobacter is also found in unchlorinated water but can be eliminated by boiling.
The family of Norwalk-like viruses (NLVs) is spread through feces-contaminated water, and are thus largely associated with shellfish. Although most people have been exposed to NLVs at some point in their lives, they rarely exhibit any symptoms. Disease associated with infection is usually mild, with symptoms of vomiting, nausea, and abdominal pain, but infection is usually self-limiting and symptoms subside within 2 days.
Corporations and government health officials have put much effort into food testing to prevent human suffering and avoid lawsuits. The two big challenges to large-scale testing of samples, both during food processing and from a clinical perspective, are time and sensitivity. Traditional microbiological methods require that the microbes be cultured and characterized for a variety of metabolic and physical markers. This process can take days to weeks, depending on the organism. For example, in a test to detect Salmonella, one bacterial pre-enrichment step takes 16–20 h, a Salmonella-specific enrichment takes another 24 h, and a final identification step in which cultures are streaked out onto selective media can take 24–48 h. If the results are positive, they must then be confirmed by sub-cultivation and serological testing. Thus, this assay can take anywhere from 3 to 6 days. During this time, the food products might decay beyond the selling point. Consumers might contract food poisoning from the products, putting them at risk for sickness or death. It is therefore preferable to have an assay that can locate and identify the offending microbes in a timeframe that is measured in hours, not days.
Similarly, the assay must also be very sensitive because the tested samples might have no more (and possibly less) than one cell per milliliter or gram of starting material. Listeria infections can start from as few as 10 cells. As with most traditional microbiological tests, this requires some form of microbe culture enrichment using a growth medium, but it is critical that this step not take too much time (see, for example, pubs.acs.org/subscribe/journals/tcaw/12/i03/pdf/303willis.pdf).
Bacteria and their enzymes, along with some fungi and critical nutrient additives are cost effective agents for in-situ remediation (otherwise known as bioremediation) of hazardous wastes and subsurface pollution in soils, sediments and wastewaters. The ability of each bacterial strain to degrade toxic waste depends on the nature of each contaminant. Since most sites are typically comprised of multiple pollutant types, the most effective approach to bioremediation is to use a mixture of bacterial species/strains, each specific to the degradation of one or more types of contaminants. It is critical to monitor the composition of the indigenous and added bacterial consortium in order to evaluate the activity level of the bacteria, and to permit modifications of the nutrients and other conditions for optimizing the bioremediation process. Additionally, it is desirable to return a bioremediation site to its natural state following the bioremediation process. Thus, monitoring of levels of bioremediating bacteria becomes necessary in assessment of the return of the site to the natural state. Fungal bioremediation is also possible. This technology utilizes white-rot fungi to clean up a wide spectrum of soil pollutants, such as wood preservatives, polycyclic aromatic hydrocarbons, organochlorines, polychlorinated biphyenyls, dyes, pesticides, fungicides, herbicides, and others. Rapid throughput methods for detection and identification of the members of the indigenous and added bacterial and/or fungal consortium would greatly facilitate characterization of such bioremediative processes.
Household mold, is a growing concern for homeowners. Molds not only pose serious threats to a home's construction and survival, they pose serious threats to one's health. More than 100,000 types of molds have been discovered yet little is known about the life of these molds and their allergens, and how they become airborne.
Stachybotrys is a species of mold which has earned the title “toxic black mold,” as it is one of the most lethal, yet common forms. Stachybotrys can become airborne and cause serious respiratory difficulties, memory and hearing loss, hemorrhaging, dizziness and sometimes death. Prolonged exposure to this strain can impair memory.
Cladosporium, Penicillium and Alternaria are more frequently detected in household mold problems. While not as likely to pose a fatal threat, these molds are known for causing asthma-related symptoms. Studies suggest that such molds are culpable for, or at least connected to, the tripled asthma rate in the past 20 years (see, for example, www.paloaltoonline.com/paw/paonline/news_features/real_estate/spring2002/2002—03—13.m old.shtml).
Identification of species of molds would also benefit from a rapid method for detection and identification.
Mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated. Low-resolution MS may be unreliable when used to detect some known agents, if their spectral lines are sufficiently weak or sufficiently close to those from other living organisms in the sample. DNA chips with specific probes can only determine the presence or absence of specifically anticipated organisms. Because there are hundreds of thousands of species of benign bacteria, some very similar in sequence to threat organisms, even arrays with 10,000 probes lack the breadth needed to detect a particular organism.
Antibodies face more severe diversity limitations than arrays. If antibodies are designed against highly conserved targets to increase diversity, the false alarm problem will dominate, again because threat organisms are very similar to benign ones. Antibodies are only capable of detecting known agents in relatively uncluttered environments.
Several groups have described detection of PCR products using high resolution electrospray ionization-Fourier transform-ion cyclotron resonance mass spectrometry (ESI-FT-ICR MS). Accurate measurement of exact mass combined with knowledge of the number of at least one nucleotide allowed calculation of the total base composition for PCR duplex products of approximately 100 base pairs. (Aaserud et al., J. Am. Soc. Mass Spec., 1996, 7, 1266–1269; Muddiman et al., Anal. Chem., 1997, 69, 1543–1549; Wunschel et al., Anal. Chem., 1998, 70, 1203–1207; Muddiman et al., Rev. Anal. Chem., 1998, 17, 1–68). Electrospray ionization-Fourier transform-ion cyclotron resistance (ESI-FT-ICR) MS may be used to determine the mass of double-stranded, 500 base-pair PCR products via the average molecular mass (Hurst et al., Rapid Commun. Mass Spec. 1996, 10, 377–382). The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for characterization of PCR products has been described. (Muddiman et al., Rapid Commun. Mass Spec., 1999, 13, 1201–1204). However, the degradation of DNAs over about 75 nucleotides observed with MALDI limited the utility of this method.
U.S. Pat. No. 5,849,492 describes a method for retrieval of phylogenetically informative DNA sequences which comprise searching for a highly divergent segment of genomic DNA surrounded by two highly conserved segments, designing the universal primers for PCR amplification of the highly divergent region, amplifying the genomic DNA by PCR technique using universal primers, and then sequencing the gene to determine the identity of the organism.
U.S. Pat. No. 5,965,363 discloses methods for screening nucleic acids for polymorphisms by analyzing amplified target nucleic acids using mass spectrometric techniques and to procedures for improving mass resolution and mass accuracy of these methods.
WO 99/14375 describes methods, PCR primers and kits for use in analyzing preselected DNA tandem nucleotide repeat alleles by mass spectrometry.
WO 98/12355 discloses methods of determining the mass of a target nucleic acid by mass spectrometric analysis, by cleaving the target nucleic acid to reduce its length, making the target single-stranded and using MS to determine the mass of the single-stranded shortened target. Also disclosed are methods of preparing a double-stranded target nucleic acid for MS analysis comprising amplification of the target nucleic acid, binding one of the strands to a solid support, releasing the second strand and then releasing the first strand which is then analyzed by MS. Kits for target nucleic acid preparation are also provided.
PCT W097/33000 discloses methods for detecting mutations in a target nucleic acid by nonrandomly fragmenting the target into a set of single-stranded nonrandom length fragments and determining their masses by MS.
U.S. Pat. No. 5,605,798 describes a fast and highly accurate mass spectrometer-based process for detecting the presence of a particular nucleic acid in a biological sample for diagnostic purposes.
WO 98/21066 describes processes for determining the sequence of a particular target nucleic acid by mass spectrometry. Processes for detecting a target nucleic acid present in a biological sample by PCR amplification and mass spectrometry detection are disclosed, as are methods for detecting a target nucleic acid in a sample by amplifying the target with primers that contain restriction sites and tags, extending and cleaving the amplified nucleic acid, and detecting the presence of extended product, wherein the presence of a DNA fragment of a mass different from wild-type is indicative of a mutation. Methods of sequencing a nucleic acid via mass spectrometry methods are also described.
WO 97/37041, WO 99/31278 and U.S. Pat. No. 5,547,835 describe methods of sequencing nucleic acids using mass spectrometry. U.S. Pat. Nos. 5,622,824, 5,872,003 and 5,691,141 describe methods, systems and kits for exonuclease-mediated mass spectrometric sequencing.
Thus, there is a need for a method for bioagent detection and identification which is both specific and rapid, and in which no nucleic acid sequencing is required. The present invention addresses this need.